1. Field of the Invention
The present invention relates to cyclic peptides that bind to the cell surface receptor (uPAR) for urokinase plasminogen activator (uPA) and, thus, are capable of delivering therapeutic agents or diagnostic probes to the surfaces of cells expressing this receptor. The invention also relates to pharmaceutical compositions comprising these peptides and their use to inhibit the binding of uPA to its cell surface receptor. By targeting therapeutic agents to uPAR or by inhibiting the binding of uPA to uPAR, it is possible to achieve a number of biological effects that include cell death, the inhibition of cell movement and migration and the inhibition of angiogenesis.
The peptides of the invention are capable of carrying a suitable detectable or imageable label so that they can be used to quantitate uPAR levels in vitro and in vivo. Such compositions are therefore useful as diagnostic, prognostic and imaging tools in all diseases and conditions where this receptor plays a pathological or otherwise undesirable role.
The peptides of the invention can also be immobilized to a suitable matrix and can be used for research applications to identify and isolate cells expressing uPAR and to identify and isolate uPAR from biological samples.
2. Description of the Background Art
The urokinase -type plasminogen activator (uPA) system is strongly linked to pathological processes, such as cell invasion and metastasis in cancer (Dan.phi. et al., Adv. Cancer Res., 44:139-266 (1985)). Cells produce uPA in an inactive form, pro-uPA or single-chain uPA (scuPA), which then binds to its receptor, uPAR. This binding event is a prerequisite for the efficient activation of scuPA to two-chain uPA (tcuPA) in a cell milieu (Ellis et al., J Biol. Chem., 264:2185-88 (1989)).
The amino acid sequence of the N-terminus of human pro-uPA [residues 1-44 of SEQ ID NO:1] is
Ser Asn Glu Leu His Gln Val Pro Ser Asn Cys Asp Cys Leu Asn Gly 1 10 PA1 Gly Thr Cys Val Ser Asn Lys Tyr Phe Ser Asn Ile His Trp Cys Asn Cys 20 30 PA1 Pro Lys Lys Phe Gly Gly Gln His Cys Glu Ile 40 PA1 X.sup.1 is Val, Cys, HomoCys, Glu, Asp, GluR.sup.1, AspR.sup.1, or Ala; PA1 X.sup.2 is Ser, Cys, HomoCys, Glu, Asp, GluR.sup.1, AspR.sup.1, or Ala; PA1 X.sup.3 is Asn or Gln; PA1 X.sup.4 is Lys, Arg or His; PA1 X.sup.5 is Tyr, Trp, Phe, substituted Phe, di-substituted Phe, HomoPhenylalanine ("HomoPhe"), .beta.-(3-pyridyl)alanine, .beta.-(2-thienyl)alanine, .beta.-(1-naphthyl)-alanine, or .beta.-(2-naphthyl)alanine; PA1 X.sup.6 is Phe, Tyr, Trp, substituted Phe, di-substituted Phe, HomoPhe, .beta.-(3-pyridyl)alanine, .beta.-(2-thienyl)alanine, .beta.-(1-naphthyl)alanine, or .beta.-(2-naphthyl)alanine; PA1 X.sup.7 is Ser, Cys, HomoCys, Glu, Asp, GluR.sup.1, AspR.sup.1 or Ala; PA1 X.sup.8 is Asn, Cys, HomoCys, Glu, Asp, GluR.sup.1, AspR.sup.1 or Ala; PA1 X.sup.9 is Ile, Leu, Val, NorVal or NorLeu; PA1 X.sup.10 is His, Cys, HomoCys, Glu, Asp, GluR.sup.1, AspR.sup.1 or Ala; PA1 X.sup.11 is Trp, Tyr, Phe, substituted Phe, di-substituted Phe, HomoPhe, .beta.-(3-pyridyl)alanine, .beta.-(2-thienyl)alanine, .beta.-(1-naphthyl)alanine, or .beta.-(2-naphthyl)alanine. PA1 L1 --CO--CH.sub.2 --NH--CO--CH.sub.2 --CH.sub.2 --CH(CO--NH--CH.sub.2 --CO--NH.sub.2)--NH-- PA1 L2 --CO--CH.sub.2 --NH--CO--CH.sub.2 --CH.sub.2 --CH(CO--NH--CH(CH.sub.2 SH)--CO--NH.sub.2)--NH-- PA1 L3 --CO--CH(CH.sub.2 SH)--NH--CO--CH.sub.2 --CH.sub.2 --CH(CO--NH--CH.sub.2 --CONH.sub.2)--NH-- PA1 L4 --CO--CH.sub.2 --NH--CO--CH.sub.2 --CH.sub.2 --CH(CO--NH--CH(CH.sub.2 CH.sub.2 SH)--CO--NH.sub.2)--NH-- PA1 L5 --CO--CH(CH.sub.2 CH.sub.2 SH)--NH--CO--CH.sub.2 --CH.sub.2 --CH(CO--NH--CH.sub.2 --CONH.sub.2)--NH-- PA1 L6 --CO--CH(CH.sub.2 CH.sub.2 COR.sup.1)--NH--CO--CH.sub.2 --CH.sub.2 --CH(CO--NH--CH.sub.2 --CONH.sub.2)--NH-- PA1 L7 --CO--CH(CH.sub.2 COR.sup.1)--NH--CO--CH.sub.2 --CH.sub.2 --CH(CO--NH--CH.sub.2 --CONH.sub.2)--NH-- PA1 L8 --CO--CH.sub.2 --NH--CO--CH.sub.2 --CH.sub.2 --CH(CO--NH--CH(CH.sub.2 CH.sub.2 COR.sup.1)--CO--NH.sub.2)--NH-- PA1 L9 --CO--CH.sub.2 --NH--CO--CH.sub.2 --CH.sub.2 --CH(CO--NH--CH(CH.sub.2 COR.sup.1)--CO--NH.sub.2)--NH-- PA1 L10 --CO--CH.sub.2 --NH--CO--CH.sub.2 --CH.sub.2 --CH(CO--NH--CH.sub.2 --COR.sup.1)--NH-- PA1 L11 --CO--CH(CH.sub.2 CH.sub.2 COOH)--NH--CO--CH.sub.2 --CH.sub.2 --CH(CO--NH--CH.sub.2 --CONH.sub.2)--NH-- PA1 L12 --CO--CH(CH.sub.2 COOH)--NH--CO--CH.sub.2 --CH.sub.2 --CH(CO--NH--CH.sub.2 --CONH.sub.2)--NH-- PA1 L13 --CO--CH.sub.2 --NH--CO--CH.sub.2 --CH.sub.2 --CH(CO--NH--CH(CH.sub.2 CH.sub.2 COOH)--CO--NH.sub.2)--NH-- PA1 L14 --CO--CH.sub.2 --NH--CO--CH.sub.2 --CH.sub.2 --CH(CO--NH--CH(CH.sub.2 COOH)--CO--NH.sub.2)--NH-- PA1 (a) contacting the cell, tissue, organ or biological sample with the diagnostic composition above and (b) detecting the presence of the label associated with the cell, tissue, organ or sample.
The structure of pro-uPA [SEQ ID NO:1] is shown in FIG. 1.
uPA is a three-domain protein comprising (1) an N-terminal epidermal growth factor-like domain, (2) a kringle domain, and (3) a C-terminal serine protease domain. uPAR, the receptor for pro-uPA, is also a multi-domain protein anchored by a glycosylphosphatidylinositol anchor to the outer leaf of the cell membrane (Behrendt et al., Biol. Chem. Hoppe-Seyler, 376:269-279 (1995)). The binding of uPA to uPAR initiates two separate events: the first, extracellular proteolysis, is mediated through the activation of plasminogen to plasmin, a broad-spectrum protease which can itself activate matrix metalloprotease (MMP) zymogens (Mazzieri et al., EMBO J., 16: 2319-32 (1997)), release latent growth factors such as TGF-.beta., IGF-I, and bFGF from their binding proteins or from their binding sites within the extracellular matrix (ECM) (Falcone et al., J Biol. Chem., 268(16): 11951-11958 (1993); Lamarre et al., Biochem J, 302: 199-205 (1994); Remacle-Bonnet et al., Int. J Cancer 72:835-843 (1997)), and directly remodel certain ECM components such as fibronectin and vitronectin (Wachtfogel et al., J. Clin. Invest., 81:1310-1316 (1988); Sordat et al., Invasion Metastasis 14: 223-33 (1994).
The second series of events, triggered by uPA binding to uPAR depends upon transmembrane signal transduction and leads to the stimulation of cell differentiation and motility in several cell types, most notably endothelial cells, epithelial cells and leukocytes (Nusrat et al., Fibrinolysis 6 (suppl 1):71-76 (1992); Fazioli et al., EMBO J. 16: 7279-86 (1997); Schnaper et al., J. Cell. Physiol. 165:107-118 (1994)). This second activity is independent of the proteolytic cascade described above. uPAR mediates these signaling events despite its lack of a transmembrane domain presumably through an adaptor protein(s) which couples extracellular binding to intracellular signaling cascades . The signaling mediated by uPAR probably involves multiple pathways, as with other cytokines. Jak/STAT and MAP-dependent pathways (which overlap with Jak/STAT) have been implicated (Koshelnick et al., J. Biol. Chem. 272:28563-28567 (1997); Tang et al., J. Biol. Chem. 273:18268-18272 (1998); Dumler et al, J. Biol. Chem. 273:315-321 (1998)).
uPAR is not normally expressed at detectable levels on quiescent cells and must therefore be upregulated before it can initiate the activities of the uPA system. uPAR expression is stimulated in vitro by differentiating agents such as phorbol esters (Lund et al., J. Biol. Chem. 266:5177-5181 (1991)), by the transformation of epithelial cells, and by various growth factors and cytokines such as VEGF, bFGF, HGF, IL-1, TNF.alpha., (in endothelial cells) and GM-CSF (in macrophages) (Mignatti et al., J. Cell Biol. 113:1193-1201 (1991); Mandriota et al., J. Biol. Chem. 270:9709-9716; Yoshida et al., Inflammation 20:319-326 (1996)). This up-regulation has the functional consequence of increasing cell motility, invasion, and adhesion (Mandriota et al., supra). More importantly, uPAR appears to be up-regulated in vivo in most human carcinomas examined to date, specifically, in the tumor cells themselves, in tumor-associated endothelial cells undergoing angiogenesis and in macrophages (Pyke et al., Cancer Res. 53:1911-15 (1993) which may participate in the induction of tumor angiogenesis (Lewis et al., J. Leukoc. Biol. 57:747-751 (1995)). uPAR expression in cancer patients is present in advanced disease and has been correlated with a poor prognosis in numerous human carcinomas (Hofinann et al., Cancer 78:487-92 (1996); Heiss et al., Nature Med. 1:1035-39 (1995). Moreover, uPAR is not expressed uniformly throughout a tumor but tends to be associated with the invasive margin and is considered to represent a phenotypic marker of metastasis in human gastric cancer. The fact that uPAR expression is up-regulated only in pathological states involving ECM remodeling and cell motility such as cancer makes it an attractive marker for diagnosis as well as a selective target for therapy.
In order to design the peptides of the present invention, it was necessary first to identify the minimal binding epitope of uPA for uPAR. It had been shown earlier that the amino terminal fragment of uPA (residues 1-135) that lacked the serine protease domain, sufficed for high affinity (sub-nanomolar) binding. (Stoppelli et al., Proc. Natl. Acad. Sci. USA 82:4939-43 (1985). Subsequent work showed that the growth factor domain alone (residues 1-48) conferred this binding. (Robbiati et al., Fibrinolysis, 4:53-60 (1990); Stratton-Thomas et al., Protein Engineering 8:463-470 (1995.))
Dan.phi. et al., WO 90/12091 (Oct. 18, 1990), disclosed that the binding of uPA to uPAR could be prevented by administering a substance comprising a sequence identical or substantially identical to a uPAR binding site of uPA amino residues 12-32. Rosenberg et al., WO 94/28145(Dec. 9, 1994) disclosed the preparation and use of non-fucosylated HuPA.sub.1-48 that prevented uPA binding to uPAR.
Earlier studies with peptide fragments within the growth factor domain had shown that residues 20-30 conferred the specificity of binding, but that residues 13-19 were also needed if residues 20-30 were to attain the proper binding conformation. Specifically, the peptide [Ala.sup.19 ]uPA(12-32), which contains two cysteines (the third cysteine being replaced by Ala to avoid undesired disulfide bond formations) in its open chain form prevented uPA binding to uPAR with an IC.sub.50 of 100 nM. In its oxidized cyclic form with an intrachain disulfide bond between Cys.sup.13 and Cys.sup.31, the peptide prevented uPA binding with an IC.sub.50 of 40 nM. The authors proposed that residues 13-19 might act indirectly to provide a scaffold that would help residues 20-30 attain the correct binding conformation (Appella et al., J. Biol. Chem., 262:4437-4440 (1987).
These results were partially confirmed by Kobayashi et al. (Int. J. Cancer, 57:727-733 (1994)) who reported that, while the linear peptide 20-30 inhibited the binding of uPA to uPAR with an IC.sub.50 of 1,000 nM, the longer peptide 17-34 was significantly more potent (IC.sub.50 =100 nM). The corresponding longer peptide (17-34) derived from the mouse sequence inhibited spontaneous metastasis of Lewis Lung carcinoma in mice, whereas the corresponding linear shorter peptide (20-30) did not.
Most recently, Magdolen et al., Eur. J. Biochem., 237:743-751 (1996) reported results of alanine-scanning mutagenesis of the binding loop of the N-terminal uPA fragment and showed that the side chains of Asn22, Lys23, Tyr24, Phe25, Ile28 and Trp30 were important and should be preserved. These authors (citing Hansen et al., Biochemistry, 33:4847-64 (1994)), disclosed that the region between Thr18 and Asn32 consisted of a flexible, seven-residue omega loop that is forced into a ring-like structure. In uPA, although Cys19 and Cys31 are in close proximity to each other (0.61 nm), they do not form a disulfide bond with each other. Instead Cys19 bonds with Cys 11, and Cys31 bonds with Cys 13. See FIG. 2. Accordingly, the uPAR binding site of uPA does not form a simple, small ring structure.
In a related, commonly assigned patent application (U.S. Ser. No. 08/47,915, now U.S. Pat. No. 5,542,492 incorporated herein by reference in its entirety) Jones et al. showed that novel cyclic molecules derived from the uPA peptide fragment 20-30 (in which residue 20 is covalently bonded to residue 30) bind to uPAR with IC.sub.50 values in the 10-100 nM range.
Citation of the above documents is not intended as an admission that any of the foregoing is pertinent prior art. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicant and does not constitute any admission as to the correctness of the dates or contents of these documents.